Recognition of a virus-encoded ligand by a natural killer cell activation receptor.

Abstract

Natural killer (NK) cells express inhibitory and activation receptors that recognize MHC class I-like molecules on target cells. These receptors may be involved in the critical role of NK cells in controlling initial phases of certain viral infections. Indeed, the Ly49H NK cell activation receptor confers in vivo genetic resistance to murine cytomegalovirus (MCMV) infections, but its ligand was previously unknown. Herein, we use heterologous reporter cells to demonstrate that Ly49H recognizes MCMV-infected cells and a ligand encoded by MCMV itself. Exploiting a bioinformatics approach to the MCMV genome, we find at least 11 ORFs for molecules with previously unrecognized features of predicted MHC-like folds and limited MHC sequence homology. We identify one of these, m157, as the ligand for Ly49H. m157 triggers Ly49H-mediated cytotoxicity, and cytokine and chemokine production by freshly isolated NK cells. We hypothesize that the other ORFs with predicted MHC-like folds may be involved in immune evasion or interactions with other NK cell receptors.

Full text

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Recognition of a virus-encoded ligand by a natural
killer cell activation receptor
Hamish R. C. Smith*†, Jonathan W. Heusel*†, Indira K. Mehta*, Sungjin Kim*, Brigitte G. Dorner*, Olga V. Naidenko‡,
Koho Iizuka*, Hiroshi Furukawa*, Diana L. Beckman*, Jeanette T. Pingel*, Anthony A. Scalzo§, Daved H. Fremont‡,
and Wayne M. Yokoyama*¶
*Howard Hughes Medical Institute, and Division of Rheumatology, Department of Medicine, and‡Department of Pathology and Immunology, Washington
University School of Medicine, and Barnes-Jewish Hospital, 660 South Euclid Avenue, St. Louis, MO 63110; and§Department of Microbiology,
University of Western Australia, Queen Elizabeth II Medical Centre, Nedlands 6907, Australia
Communicated by Emil R. Unanue, Washington University School of Medicine, St. Louis, MO, April 29, 2002 (received for review April 19, 2002)
Natural killer (NK) cells express inhibitory and activation receptors
that recognize MHC class I-like molecules on target cells. These
receptors may be involved in the critical role of NK cells in
controlling initial phases of certain viral infections. Indeed, the
Ly49H NK cell activation receptor confers in vivo genetic resistance
to murine cytomegalovirus (MCMV) infections, but its ligand was
previously unknown. Herein, we use heterologous reporter cells to
demonstrate that Ly49H recognizes MCMV-infected cells and a
ligand encoded by MCMV itself. Exploiting a bioinformatics ap-
proach to the MCMV genome, we find at least 11 ORFs for
molecules with previously unrecognized features of predicted
MHC-like folds and limited MHC sequence homology. We identify
one of these, m157, as the ligand for Ly49H. m157 triggers Ly49H-
mediated cytotoxicity, and cytokine and chemokine production by
freshly isolated NK cells. We hypothesize that the other ORFs with
predicted MHC-like folds may be involved in immune evasion or
interactions with other NK cell receptors.
V
decreased MHC class I expression may enhance susceptibility to
natural killer (NK) cells (2) that are normally prevented from
killing by MHC class I-specific inhibitory receptors (3, 4).
Herpesviruses counter with molecules that engage inhibitory
receptors (5–7). Because inhibitory NK cell receptors block NK
cell function by preventing signaling through activation recep-
tors, this viral strategy suggested that NK cell activation recep-
tors are critical to control virus infection.
One such NK cell activation receptor was recently revealed
after a combination of genetic and immunological approaches.
C57BL?6 mice are resistant to murine cytomegalovirus
(MCMV), as manifested by splenic control of viral replication
and survival, whereas susceptible strains (BALB?c and DBA?2)
display high splenic viral titers and lethality. This phenotypic
difference is controlled by an autosomal dominant locus termed
Cmv1 (8) that was mapped to the NK gene complex (NKC) on
distal mouse chromosome 6 (9) that contains gene clusters for
NK cell receptors. Consistent with NK cell-mediated resistance,
mAb elimination of NK cells converts resistance to susceptibility
(10). Detailed genetic and physical mapping yielded an infor-
mative recombinant inbred mouse strain, BXD-8, that retains
the resistant NKC haplotype but was susceptible. BXD-8 has a
selective deletion in the gene for Ly49H, a putative NK cell
activation receptor (11, 12). Furthermore, when Ly49H was
perturbed by specific mAb, MCMV replication was uncon-
trolled, leading to lethality in otherwise resistant mice (11–13).
These genetic and immunologic data provided strong evidence
that Ly49H is required for NK cell-mediated resistance to
MCMV infection.
Ly49HisamemberoftheLy49familyofNKcellreceptorsand
is expressed on about 50% of NK cells in C57BL?6 mice (14–16).
Unlike the original Ly49 family member, the MHC class
I-specific inhibitory receptor Ly49A, Ly49H lacks cytoplasmic
iruses have evolved several means to down-regulate MHC
class I expression to evade specific immunity (1). However,
immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and
instead contains a charged transmembrane residue that permits
association with the immunoreceptor tyrosine-based activation
motif (ITAM)-containing DAP12 [also known as killer cell-
activating receptor-associated protein (KARAP)] molecule.
Previous in vitro studies have demonstrated that cross-linking of
Ly49H results in tyrosine phosphorylation of DAP12?KARAP
and downstream activation events, including cytokine produc-
tion and cytotoxicity (14, 15). However, in vivo studies revealed
that there was no preferential activation of Ly49H?NK cells
soon after MCMV infection (17). Instead, there is first an early
(day 1–2) ‘‘nonspecific’’ phase characterized by IFN-? produc-
tion and proliferation of NK cells, without regard to Ly49H
expression, that could be due to global activation by systemic
cytokines(18).Itisnotuntilsomewhatlater(day4)thatselective
activation of Ly49H?NK cells could be recognized, i.e., a
‘‘specific’’ phase in which there is preferential proliferation of
Ly49H?NK cells, and this proliferation is blocked by the
anti-Ly49H mAb. These findings appeared to be discordant with
the observation that NK cells from resistant mice are able to
control MCMV replication in the early phase of infection,
suggesting that the ‘‘nonspecific’’ early phase may be masking
detection of specific activation of Ly49H?NK cells by MCMV.
The previous in vivo analysis also suggested that Ly49H
specifically recognizes MCMV-infected cells. Because the
known ligands for Ly49 receptors are MHC class I molecules and
other NKC-encoded molecules (NKG2D, CD94-NKG2) can
recognizeMHCclassI-likestructures(19),theLy49Hligandwas
speculated to be an MHC-related molecule of host (11) or virus
(11, 12) origin, although other host or viral molecules remained
possible (11). The ligand may be constitutively expressed on
normal cells, but only on virus-mediated down-regulation of
MHC class I expression would NK cells be released from
inhibition and kill the target, as predicted by the ‘‘missing-self’’
hypothesis (2). Alternatively, perhaps ligand expression could be
induced, as for NKG2D ligands (19). Therefore, another issue in
understanding innate NK cell control of viral infection was the
nature of the ligand for Ly49H.
Herein, we used heterologous reporter cells to demonstrate
that Ly49H is triggered by MCMV-infected cells. We examined
the basic parameters of the putative ligand, including specificity
and, by excluding most of the other possibilities, reached the
interim conclusion that the ligand is encoded by the virus itself.
By exploiting a bioinformatics approach, we determined that
MCMV has at least 12 ORFs encoding molecules with putative
Abbreviations:NK,naturalkiller;MCMV,murinecytomegalovirus;NKC,NKgenecomplex;
ITAM, immunoreceptor tyrosine-based activation motif; KARAP, killer cell-activating re-
ceptor-associated protein; ?-gal, ?-galactosidase; X-Gal, 5-bromo-4-chloro-3-indolyl ?-D-
galactoside;GFP,greenfluorescentprotein;CHO,Chinesehamsterovary;ATAC,activation-
induced T cell-derived and chemokine-related cytokine; BM, bone marrow.
†H.R.C.S. and J.W.H. contributed equally to this work.
¶To whom reprint requests should be addressed. E-mail: yokoyama@imgate.wustl.edu.
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MHC class I-like folds. A systematic examination of these and
othercandidatemoleculesledtotheidentificationofm157asthe
ligand for Ly49H. m157-transfected target cells are selectively
killed by Ly49H?NK cells and can quickly stimulate the
activation of freshly isolated NK cells, suggesting that specific
activation of Ly49H?NK cells early in infection is masked
in vivo.
Methods
Cells and Generation of BWZ Transfectants. All cells and media are
described in detail in supporting text (which is published as
supporting information on the PNAS web site, www.pnas.org).
The BWZ.36 cell line, generously provided by N. Shastri (Uni-
versity of California, Berkeley, CA) (20) was cultured in RPMI
1640 containing 10% FCS and 200 units?ml hygromycin (Cal-
biochem). The pMX, pMX-puro (puromycin-resistant) or pMX-
internal ribosomal entry site (IRES) plasmid vectors (21), and
the PLAT-E packaging cell line were generously provided by T.
Kitamura, (University of Tokyo).
Reporter Assay with MCMV-Infected Cells. Cells were infected with
MCMV at a multiplicity of infection of 5 and agitated every 10
min for 60 min. ?HV68 and HSV infections were performed
similarly except in media containing 2% FCS and with 2 h
adsorption time. When indicated, antibodies were added at a
final concentration of 20 to 50 ?g?ml, and mAb 2.4G2 (20
?g?ml) was added to prevent inadvertent Fc receptor mediated
effects. For UV radiation-mediated inactivation, an aliquot of
MCMVwasexposedtoa30-WgermicidalUVlampatadistance
of ?7 cm for 30 min with agitation. The titer of the UV-
inactivated virus was ?102plaque-forming units?ml (limit of
detection) in a plaque assay with 3T12 fibroblasts. After
coculture with MCMV-infected or m157-transfected cells,
?-galactosidase (?-gal) activity was quantitatively assayed in
BWZ transfectants with the substrate chlorophenol red ?-D-
galactoside (CPRG; Calbiochem) or qualitatively by fixation and
staining with 5-bromo-4-chloro-3-indolyl ?-D-galactoside (X-
Gal) as described (20). Photomicrographs were acquired by
using a Nikon TMS-F microscope and a Nikon model 990 digital
camera.
Flow Cytometric Analysis and Intracellular Staining. For routine
staining, cells were analyzed with a FACSCalibur instrument
(BD Biosciences) as previously described (15). Data from 104to
105gated events were collected. For intracellular staining,
freshly isolated B6.RAG1?/?splenocytes were cocultured for
6–8 h with uninfected or MCMV-infected IC-21 cells (1:1 ratio)
in a 96-well plate (150,000 cells each). In a different experiment,
we cocultured B6.RAG1?/?splenocytes with BaF3 transfectants
(1:1 ratio). In all experiments, brefeldin A was added for the last
5–7 h. Cells were stained with anti-NK1.1-APC, anti-CD3-
PerCP-Cy5.5 (PharMingen), and anti-Ly49H-biotin (clone
3D10), along with unlabeled mAb 2.4G2 to block Fc receptor
binding,followedbystreptavidin-phycoerythrin.Cellswerefixed
andpermeabilizedwiththeCytofix?Cytopermkit(PharMingen)
accordingtothemanufacturer’sinstructions.IntracellularIFN-?
was detected with XMG1.2-FITC (PharMingen) and intracellu-
lar activation-induced T cell-derived and chemokine-related
cytokine (ATAC)?lymphotactin with mAb MTAC-2 (22). Cells
were gated on the NK1.1?CD3?population.
Cloning of Ly49H Ligand. PCR primers were designed to amplify
each candidate ORF from 3 nt before and after the predicted
ORF and contained restriction enzyme sites for directional
cloning into the pMX-puro vector. ThermalAce DNA polymer-
ase (Invitrogen) or Pfu DNA polymerase (Stratagene) were used
to PCR amplify a cDNA library produced from MCMV-infected
IC-21 cells. Readily generated amplicons were ligated into
pMX-puro vector. To neutralize possible PCR errors, five inde-
pendent clones of each amplicon were isolated, pooled, and used
as a group to transfect PLAT-E cells. ORF transfectants were
used to stimulate BWZ transfectants or generate retroviruses for
infection of target cells. Puromycin-resistant cells expressing
each ORF were cocultured with HD12 cells for 12–22 h, which
were then fixed and stained with X-GAL. The sequence of
multiple m157 clones was verified.
IL-2-Activated NK (LAK) and Cytotoxicity Assays.GenerationofLAK
cells, sorted Ly49H?and Ly49H?NK cells, and standard 4 h
51Cr-release assays were performed as previously described (15,
23). Alexa 488-labeled Fab fragments of mAb 3D10 were used
for flow sorting of Ly49H subsets.
Results
Specific Activation of Ly49H by MCMV-Infected Cells. To test the
hypothesis that Ly49H specifically recognizes MCMV-infected
cells, we expressed Ly49H with DAP12?KARAP (referred to as
HD12) or other control constructs in heterologous BWZ.36 cells
(20), containing a stably integrated reporter construct for ?-gal
that is activated on ITAM signaling (Fig. 1A). As with T cell
antigen receptor (TCR) stimulation in other BWZ transfectants
(20), immobilized anti-Ly49H led to ?-gal expression in HD12
cells but not in BWZ transfected with vector, green fluorescent
protein (GFP), or DAP12?KARAP alone as indicated by ?-gal
substrates (data not shown). A related DAP12?KARAP-
associated NK cell activation receptor, Ly49D, specifically rec-
ognizes Chinese hamster ovary (CHO) cells (24) but does not
participate in MCMV immunity (11). Ly49D plus DAP12?
KARAP transfectants (DD12) were not stimulated by anti-
Ly49H (data not shown). Conversely, DD12 cells showed strong
?-gal induction when stimulated with CHO cells (Fig. 1B,C),
consistent with Ly49D ligand recognition, and suggesting that
HD12 cells could be used as reporters to test ligand recognition
by Ly49H.
Indeed, MCMV but not mock-infected macrophage cells
induced ?-gal activity in HD12 cells (Fig. 1B,C). Although DD12
cells expressed comparable receptor levels (see supporting in-
formation), DD12 cells were not activated by MCMV-infected
cells (Fig. 1 B and C). Furthermore, HD12 activation by MCMV-
infected stimulators depended on DAP12?KARAP signaling,
because the HD12.Y2F line, which expresses Ly49H with a
signaling-deficient mutant form of DAP12?KARAP, showed no
detectable ?-gal activity. Soluble mAbs against Ly49H or Ly49D
completely abrogated the activation of HD12 or DD12 reporter
cells by MCMV-infected or CHO stimulators, respectively, in-
dicating Ly49H (and Ly49D) specificity (Fig. 1B). Other control
mAbs had no effect (data not shown). These data indicated that
MCMV-infected cells directly and specifically activate the re-
porter cells through Ly49H, demonstrating that the ligand for
Ly49H is expressed by MCMV-infected cells.
Multiple MCMV ORFs for Putative MHC Class I Molecules. HD12 cells
were not triggered by target cells stimulated with IFN-? or
supernatants from MCMV-infected cells (see supporting infor-
mation). Fixed infected cells stimulated HD12 cells, indicating
that the ligand was expressed on the cell surface. However,
stimulation occurred even with infected macrophages from H-2
Db?Kb??2-microglobulin (?2m)-triple-deficient (3KO) mice,
against the possibility that the ligand is a host MHC class I
molecule. Infection with other herpes viruses known to infect
rodent cells (herpes simplex, ?HV68) failed to stimulate HD12
cells, implying that the ligand is MCMV specific. HD12 cells
were activated by MCMV-infected cells as little as 8 h postin-
fection (infection plus activation time). These data suggested
that the ligand for Ly49H is expressed on the surface of
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MCMV-infected cells with early phase kinetics and is most likely
encoded by the virus itself.
To identify the putative MCMV-encoded ligand for Ly49H,
we took a bioinformatics approach and examined the 230-kb
MCMV genome for candidate ORFs (25). In general, herpes-
virus genomes contain a central 100-kb region with genes closely
related to each other whereas sequences at either end are
unrelated (25–27). In MCMV, the end regions include the m02
and m145 gene families. However, we eliminated the m02 to m16
region because a mutant MCMV strain (SMsubm02-16) with a
deletion spanning this region (28) stimulated HD12 cells (data
not shown). We therefore examined the MCMV genome for
other ORFs containing probable signal peptides, N-linked gly-
cosylation sites, and hydrophobic transmembrane domains. We
were particularly interested in possible MHC class I-related
molecules, but only m144 has primary sequence homology to
MHC class I detectable by BLAST (25). Surprisingly, however, we
found that MCMV contains at least 11 other ORFs encoding
molecules with potential MHC-like folds, as determined by
structural homology analysis [3D-PSSM (29)], even though they
show little sequence relationship to known MHC class I family
members(Table1).ManyoftheseORFswereinthem145family
and became prime candidates for the putative Ly49H ligand.
Identification of m157 as the Ligand for Ly49H. PCR primers for the
m145 family and other candidate ORFs were used to amplify
cDNAs from an MCMV-infected IC-21 library. Twenty nine
readily produced amplicons (m17, m37, m42, m73, m74, m90,
m92, m100, m117.1, m124, m136, m138, m144, m145, m146,
m150, m151, m152, m153, m154, m155, m157, m158, m159,
m160, m163, m164, m165, and m167) were expressed in cell lines
with highly efficient retroviral vectors. Only transfectants ex-
pressing m157, one of the predicted MHC class I folded proteins,
stimulated HD12 cells (Fig. 2A and data not shown), and this
stimulation was blocked specifically by anti-Ly49H mAb (Fig.
2B). None of the other transduced ORFs stimulated HD12 cells
(data not shown). This list included m144, the MCMV MHC
class I-like molecule (6), previously speculated to be the ligand
for Ly49H (12). Furthermore, expression of m157 in resistant
targets conferred markedly enhanced susceptibility to lysis by
Fig. 1.
reporter assay. cDNA cassettes for murine DAP12, Ly49H, or Ly49D were cloned into the multiple cloning site (MCS) upstream from the internal ribosomal entry
site(IRES)ofpMX-IRES-GFPtogeneratethecontrolvectorsDAP12-GFP,H-GFP,andD-GFP.TheGFPcassetteinthedownstreampositionwasreplacedwitheither
a wild-type or ITAM-deficient mutant murine DAP12 cassette to generate the pMX-HD12, pMX-HD12.Y2F, pMX-DD12, and pMX-DD12.Y2F expression vectors.
IndividualconstructsweretransfectedintoBWZcells.Forexample,thepMX-HD12constructwasusedtoproduceHD12indicatorcellsinwhichLy49Hisassociated
with DAP12 (KARAP) that contains ITAMs (boxes). Activation through Ly49H and DAP12 leads to nuclear factor of activated T cells (NFAT)-induced production
of ?-gal. (B) Chlorophenol red ?-D-galactoside analysis of BWZ.36 reporter lines after coculture with mock- or MCMV-infected primary bone marrow
macrophages,asindicated.Ly49D-expressinglines(DD12andDD12.Y2F)wereadditionallytestedwithCHOcellsasindicated.ForHD12andDD12lines,theeffect
of addition of mAbs 3D10 (anti-Ly49H) or 4E4 (anti-Ly49D) are indicated (arrows). Data are expressed as a percentage of maximal ??gal induction by phorbol
12-myristate13-acetate(PMA)?ionomycinforeachreporter-stimulatorcombination.(C)X-GalstainingoftheindicatedBWZreporterlinesaftercoculturewith
mock- or MCMV-infected IC-21 cells. (Insets) Maximal ?-gal induction after stimulation with PMA ? ionomycin. The Ly49D-expressing DD12 and DD12.Y2F lines
were additionally stimulated with CHO cells, known to express a cognate ligand for Ly49D (24).
Specific activation of Ly49H-expressing reporter cells by MCMV-infected cells. (A) Schematic illustration of retroviral vector constructs and the HD12
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bulk NK cells (data not shown), and in particular to Ly49H?but
not Ly49H?NK cells (Fig. 2C). Susceptibility was specifically
reversed by the anti-Ly49H mAb (Fig. 2C). Finally, MCMV-
infected bone marrow (BM) macrophages retained susceptibility
to Ly49H?NK cells whereas they became more resistant to
Ly49H?NK cells, an effect also reversed by the anti-Ly49H mAb
(Fig. 2C). Taken together, these data indicated that Ly49H on
NK cells specifically recognizes the product of the m157 ORF.
Stimulation of Fresh NK Cells by m157. The m157 ORF encodes a
deduced 329-aa polypeptide with no sequence homology to any
other molecule in GenBank except for r157, its apparent ho-
molog in the rat CMV genome (data not shown). m157 contains
a potential N-terminal 21-aa signal peptide, and an ectodomain
with five potential sites for N-linked glycosylation. However, a
putative hydrophobic transmembrane domain is interrupted by
a stop codon. Because the related domain in r157 does not
contain a stop codon, we verified the sequence of m157 with
multiple reverse transcription–PCR primers (data not shown).
All cDNA clones contained m157 sequence identical to that in
the published annotated MCMV genome (25) and m157 has a
putative glycosylphosphatidylinositol linkage (Table 1).
Importantly, the identification of the Ly49H ligand also
permits advances in understanding of the in vivo NK cell
response to MCMV because it provided a means to examine
whether Ly49H?NK cells are selectively activated soon after
exposure to MCMV-infected cells. After only a brief coincuba-
tion of freshly isolated C57BL?6 RAG-1?/?splenocytes (devoid
of mature B and T cells) with MCMV-infected IC-21 targets in
vitro, there was selective activation of Ly49H?NK cells as
indicated by enhanced intracellular IFN-? staining, primarily in
Ly49H?NK1.1?cells (Fig. 3A). This effect was blocked by the
anti-Ly49H mAb (Fig. 3A). A similar effect was also seen with
m157-transfected BaF3 cells that was not observed with untrans-
fected or other transfected BaF3 cells (Fig. 3B) or in costaining
with anti-Ly49D (Fig. 3C). The m157 effect was also reversed
with anti-Ly49H (Fig. 3C) but not a control Ab (data not shown).
Strikingly, we also observed decreased anti-Ly49H binding in
response to m157 targets and to a lesser extent with MCMV
infection. It is also notable that the IFN-?-expressing Ly49H?
cells showed lower reactivity with anti-Ly49H, indicating a
correlation with NK cell activation. Finally, MCMV infection
and m157 also triggered Ly49H?NK cells to express a chemo-
kine, ATAC?lymphotactin (ref. 30; Fig. 3D), that may serve to
recruit or activate other inflammatory cells. Thus, m157 triggers
specific activation of primary Ly49H?NK cells, strongly sug-
gesting that m157 quickly stimulates specific NK cell activation
in vivo.
Discussion
m157 belongs to the m145 family, comprised of related glyco-
proteins (?20% amino acid identity) including m152 (25). These
proteins are generally expressed early during infection and are
dispensable for viral growth and replication in vitro (31). The
Ly49H ligand appears to be less well expressed on MCMV-
infected 3KO macrophages (see supporting information), sug-
gesting that m157 expression may be enhanced by association
with MHC class I or ?2-microglobulin molecules. In this regard,
m152 can retain MHC class I heavy chains in the endoplasmic
reticulum-Golgi intermediate compartment (32) and also has a
predicted MHC class I fold. These data suggest that m157 may
have a role in altering the MHC class I assembly and presenta-
tion pathway, perhaps in an allotype or tissue-specific manner
(33, 34). Alternatively, because it is known that individual MHC
class I ligands can bind both NK cell activation and inhibitory
receptors (3, 23, 35), m157 may also bind an inhibitory receptor
in C57BL?6 or other strains. These possible alternative immune
evasion functions for m157, the limited sequence availability of
MCMV strains, and the restricted expression of Ly49H to a
relatively small number of inbred mouse strains (9, 36) may
explain why MCMV retains this ORF in its genome. The
complexities of interactions between the various ORF products,
especially m157 and the putative inhibitory receptor ligand
Table 1. MCMV ORFs encoding proteins with predicted MHC-like folds
MCMV ORF Top hit* (MHC-fold) E-value of top hit (% confidence)
Sequence comparison to top hit†
No. of hits to MHC-folds Aligned?total Identical?similar
m17
m37
m90
m144
m145
m150
m151
m152
m153
m155
m157
m159
HLA-A2
ZAG
ZAG
HLA-B8
mCD1d1
MIC-A
T22
MIC-A
T22
mCD1d1
H2-M3
ZAG
0.35 (70)
0.118 (80)
0.027 (95)
0.00013 (95)
0.0287 (95)
0.0146 (95)
0.0299 (90)
0.03 (95)
0.0534 (90)
0.056 (90)
0.089 (90)
0.0094 (95)
151?314
170?256
153?191
229?252
245?411
246?298
235?316
286?317
235?318
244?313
225?289
240?343
31?68
29?62
13?45
59?209
30?96
45?98
39?118
31?118
36?67
39?87
50?74
44?91
3
6
4
6
7
11
6
7
5
3
7
7
*All predicted ORFs of the MCMV genome were analyzed with the 3D-PSSM search algorithm (29), which recognizes remote protein sequence homologies based
onthree-dimensionalposition-specificscoringmatrices.Thepredictedmatureectodomainsof12MCMVORFshadtopalignmenthitswithprofilesofMHCclass
I or class I-related crystal structures with statistical E-values less than unity. The E-value is the number of database hits expected by chance. Lower E-values
represent more significant scores to which 3D-PSSM assigns % confidence. The protein database (PDB) ID codes for the top hit classical and non-classical MHC
class I molecules are: HLA-A2 (1duz), HLA-B8(1 age), ZAG (1zag; zinc-?2-glycoprotein), mCD1d1 (1cd1; murine CD1d), MIC-A (1hyr), T22 (1c16), H2-M3 (1mhc).
Reported in the rightmost column is the total number of hits with E-values less than unity that are MHC-related structures for the given ORF.
†Shown is the number of viral ORF residues that were sequence aligned to the top 3D-PSSM top hit, as is the total number of residues in the mature ectodomains
that were queried. Also detailed is the number of identical residues, as well as the total number of identical and conservatively substituted residues in the
alignments. Analysis of these MCMV ORFs by BLAST produced significant MHC-fold similarities (E-values smaller than 1) only for m144, a previously identified
class I-related molecule that associates with ?2m (44). All 12 of these MHC class I-like MCMV ORFs, except m157, are predicted type I transmembrane proteins,
with a canonical N-terminal leader peptide signal sequence and short cytoplasmic tail. (Analyzed by the signal peptide and transmembrane region prediction
servers at the Center for Biological Sequence Analysis, Technical University of Denmark, www.cbs.dtu.dk.) m157, on the other hand, has a predicted
glycosylphosphatidylinositol(GPI)membranelinkagesite.[PredictionfromtheDGPIserver(availableviaProteomicsToolsonExpasyMolecularBiologyserver).]
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m144, and their effects on NK cells during infection also require
further analysis.
Interestingly, using IFN-? production as a surrogate marker of
activation, we were able to detect specific activation of freshly
explanted Ly49H?NK cells after only a brief exposure to
m157-expressingtargets.Whenextrapolatedtotheinvivosetting
by accounting for MCMV trafficking from the peritoneum to the
spleen, infection and induction of m157 expression, and produc-
tion of IFN-? by Ly49H?NK cells, one would have expected to
detect similar Ly49H?NK cells responses in vivo within the early
period after infection. In addition, viral replication is already
controlled by Ly49H?NK cells during this time. However,
during this period, there is enhanced IL-12 production (37) that
may stimulate most NK cells to produce IFN-? regardless of
whether they express Ly49H or are triggered through this
activation receptor. Thus, our current data provide additional
evidence that the specific activation of naive Ly49H?NK cells
may be masked by such systemic responses.
The m157 induction of IFN-? may nonetheless be important
in helping to control MCMV because IFN-? would be delivered
in close proximity of infected cells. By contrast, systemic pro-
duction of IFN-? by IL-12 or other cytokines may be less
effective. In addition, we also showed that a chemokine, ATAC?
lymphotactin, was also specifically produced by m157 activation
of Ly49H?NK cells, suggesting that the response to m157 may
also provide a means for NK cells to trigger other inflammatory
events, such as the attraction or activation of other immune cells.
Thus, in addition to cytotoxicity, specific activation of NK cells
may help provide local control of virus infection and lead to a
cascade of other events in innate immunity.
A notable finding in our studies is the marked decreased
reactivity of the anti-Ly49H mAb with Ly49H?NK cells that
appeared to have encountered the m157 ligand. This effect
appeared to be specific and could be due to a variety of effects,
including receptor down-regulation akin to effects seen with T
cell antigen receptor cross-linking. Alternatively, down-
regulation of Ly49H expression could be due to receptor shed-
Fig.2.
m157 transfectants. HD12 or DD12 cells were incubated with C1498 cells
transfected with m157, or control m158, as indicated. (B) Specific blockade of
m157 activation of HD12 by anti-Ly49H mAb. HD12 cells were incubated with
J774 cells transfected with GFP or m157, in the presence of anti-Ly49H or
isotype control mAb, as indicated. (Inset) Maximal ?-gal induction with
PMA ? ionomycin. (C) Ly49H?NK cells kill MCMV-infected C57BL?6 BM
macrophages and BaF3-m157 transfectants. A standard 4-h51Cr-release assay
wasperformedwithLy49HsortedLAKcellsfromC57BL?6mice.Ly49H?(filled
symbols) or Ly49H?(open symbols) LAKs were incubated with indicated
targets at various effector-to-target (E:T) ratios in media alone (squares) or
with the anti-Ly49H mAb 3D10 (diamonds) or isotype control (circles). In the
BM macrophage experiments, F(ab?)2 fragments were used to avoid redi-
rectedlysiswhereaswholeantibodieswereusedforotherpanels.Theisotype
controlforBMmacrophageexperimentswasmAbB8-24-3,andmAb9E10was
used as isotype control in the remaining experiments. The lysis of uninfected
BM macrophages is represented by a triangle.
Recognitionofm157byLy49H.(A)SpecificactivationofHD12cellsby
Fig. 3.
ATAC by fresh NK cells. Freshly isolated splenocytes from B6.RAG-1-deficient
mice were cocultured with (A) uninfected or MCMV-infected IC-21 cells, (B)
parental BaF3 cells or BaF3 cells transfected with either m157 or GFP, (C) BaF3
cells transfected with m157, and (D) BaF3 cells transfected with m157 or
MCMV-infected IC-21 cells. All cocultures were performed at effector-to-
target ratio of 1:1. Where indicated, F(ab?)2 fragments of anti-Ly49H or
anti-Ly49D were added during incubation. Six to 8 h later, cells were stained
for intracellular IFN-? (A, B, and C) or ATAC (D) after cell surface marker
staining for NK1.1 and Ly49H or Ly49D. As controls, profiles of cytokine
production by NK cells in the absence of target cells are shown (w?o target).
Gated NK1.1?cells are shown.
Interaction of Ly49H with m157 induces production of IFN-? and
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Page 6

ding, or simply blockade of Ab binding by ligand. Nevertheless,
the effect can be viewed as a specific activation event on cells
that have engaged ligand and may itself have physiological
consequences.
Another unexpected outcome of our studies was the identi-
fication of a large number of other MCMV ORFs encoding
proteins with potential MHC-like structures. Whereas unequiv-
ocal determination of their three dimensional folds will require
crystallographic studies, these findings nonetheless provide can-
didates for further immunological study. The putative MHC
class I-like molecules may well be involved in immune evasion or
detection because they are dispensable for in vitro growth and
replication. Perhaps these molecules also perturb MHC class I
assembly and antigen presentation or interact with NK cell
receptors, like m152 and m157, respectively. These findings also
suggest that other viruses, especially large DNA viruses, may
containpreviouslyunrecognizedhomologuesorfunctionalmim-
ics of NK cell receptor ligands. This result may be particularly
relevant for other viruses manifesting phenotypic resistance that
is genetically linked to the NK gene complex or other mamma-
lian genomic regions that encode NK cell receptors. Murine loci
for genetic resistance to mousepox, and herpes simplex, for
example, map to the NKC (38, 39), suggesting that they may
encode NK cell receptors that interact with viral encoded
ligands. In addition, perhaps a similar bioinformatics approach
to mammalian genomes will reveal other putative MHC-like
molecules that may serve as endogenous ligands for Ly49H and
other NK cell receptors. Thus, further analysis of these putative
MHC class I-like molecules may be revealing.
Finally, it is intriguing to note that the m145 family and m157
itself are present in the genome of rat CMV but absent in human
CMV (40). Likewise, the Ly49 gene cluster is also present in rats
(41),butonlyaremnantispresentinthehumangenome(42,43),
perhaps reflecting the coordinated evolution of pathogen im-
mune evasion and innate host response genes, a virus-host
counterpart to an international arms race.
We thank Nilabh Shastri, Toshio Kitamura, Ted Hansen, Eva-Marie
Wormstall, Pamela Stanley, David Leib, Tom Shenk, and Skip Virgin for
reagents; Marco Colonna, Leon Carayannopoulos, and Skip Virgin for
encouragement and helpful discussions; Jennifer Laurent, Kim Mar-
lotte, and Darryl Higuchi for technical assistance; and Emil Unanue,
Tony French, and Emily Ho for critical reading of the manuscript. This
work was supported by the Barnes-Jewish Hospital Foundation and
grants from the National Institutes of Health to W.M.Y., who is an
investigator of the Howard Hughes Medical Institute. A.A.S. is sup-
ported by Project Grant 990646 from the National Health and Medical
Research Council of Australia. Work in the Fremont laboratory is
supported by the ‘‘Midwest Center for Structural Genomics’’ (National
Institutes of Health?National Institute of General Medical Sciences P50
GM62414).
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